New research uncovers how the Clec14a protein in capillary endothelial cells controls bone growth and maturation, revealing a key mechanism in skeletal development.
Study: Type-H endothelial cell protein Clec14a orchestrates osteoblast activity during trabecular bone formation and patterning. Image Credit: NTshutterth/Shutterstock.com
A recent study published in Communications Biology explored the role of the C-type lectin domain containing 14A (Clec14a), a type-H endothelial protein, in coordinating osteoblast activity.
Background
The role of bone angiogenesis in regulating postnatal skeletal growth and bone remodeling and repair has received increased attention lately. Technological advances have led to the discovery of new subtypes of bone capillaries. Type-H capillaries are reported to be active modulators of osteogenesis.
Multimerin 2 (MMRN2) is an extracellular matrix protein that concomitantly binds CD248 and CLEC14A. Both regulate pathologic and embryonic angiogenesis, cell migration, and adhesion.
Previously, the authors reported that Cd248 negatively regulates osteoblast mineralization, and its deletion increases osteoblast-driven mineralization. Besides, recent reports suggest that Clec14a is upregulated during bone healing.
The study and findings
In the present study, researchers evaluated the role of Clec14a in bone formation. First, they assessed the expression of Clec14a and its ligand, Mmrn2, in the murine long bone and confirmed that both proteins were highly expressed in the bone metaphysis.
Further, Clec14a was immunolabeled in vivo using an anti-Clec14a antibody. This indicated that Clec14a predominately localizes to type-H vessel endothelium in the bone metaphysis.
Overall, the researchers confirmed that the expression of Mmrn2 and Clec14a was confined to endothelial cells (ECs), which was more pronounced in type-H vessel ECs. Next, they assessed the impact of Clec14a deletion on bone vascularization.
They observed a reduction in the percentage of type-H endothelium in Clec14a-/- mice aged two weeks relative to wild-type (WT) Clec14a+/+ mice. Capillaries in both groups increased from postnatal day 4 (P4) to four weeks.
Further, vessel density was not significantly different between the two groups. Immunofluorescence imaging of the tibia in both groups at P4 revealed a highly interconnected vascular bed with almost similar coverage of type-L and type-H capillaries.
However, in juvenile mice (aged four weeks), both groups exhibited a diminished type-H bed relative to P4 mice, distinctly localized to the superior part of the metaphysis.
Besides, at four weeks, there was a reduction in type-H column lengths and the area covered by type-H column vascular front in the metaphysis in Clec14a—/—mice.
Next, the team examined the effects of Clec14a deletion on the localization of mature and immature osteoblasts using immunostaining for type-1 collagen (Col1a1) and Osterix (Osx), respectively.
Osx+ osteoblasts were detected in high numbers in the endosteum and tibial metaphysis, regardless of the genotype, with fewer osteoblasts within the diaphysis. Clec14a-/- mice had a significantly increased number of Osx+ osteoblasts in the inferior metaphysis at P4 compared to WT mice.
Further, the team measured the tibial bone length and body weight in two-, four--, eight--, and 30-week-old WT and Clec14a-/- mice. There were no significant body weight differences at any time point. Bone elongation was the most active between two and four weeks in both genotypes when tibial length doubled. Notably, at two weeks, tibiae were shorter in WT mice than in Clec14a-/- mice.
In addition, the researchers observed embryonic day 14.5 metatarsal explant cultures over a week to understand the impact of Clec14a-/- deletion in early development.
They noted a significant increase in Clec14a—/—explant length at all time points. Next, the team used RNA sequencing of calvarial-cell isolates to better understand the molecular changes at the transcript level.
This identified 1,223 genes as differentially expressed between the two genotypes. Gene set enrichment analysis revealed robust expression of markers enriched in osteoblast proliferation and maturation and endochondral ossification pathways in Clec14a-/- samples.
Further, osteoblast maturation gene expression was examined in osteoblasts from the calvaria of WT and Clec14a-/- P4 pups cultured without ECs.
Transcript and protein levels of alkaline phosphatase (Alpl) were measured in vitro over 21 days. Alpl transcript levels increased in WT and Clec14a-/- osteoblasts in response to maturation stimuli.
However, peak Alpl expression was observed much earlier (on day 4) in Clec14a-/--isolated osteoblasts than in WT-isolated osteoblasts (on day 12). Freshly isolated passage Clec14a-/--isolated osteoblasts exhibited increased Alpl activity in response to maturation stimuli at days 6 and 8.
Conclusions
The researchers demonstrated that the expression of Clec14a and Mmrn2 is restricted to ECs in the bone stroma; both genes are highly expressed in type-H endothelium and are differentially regulated.
Findings also indicate the dysregulation of tip cell formation and migration in Clec14a-/- mice and the blunting of type-H vessel buds at the growth plate in four-week-old Clec14a-/- mice.
Overall, the study identified Clec14a as a key EC-specific target protein whose inactivation enhances bone formation.
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